The yeast two-hybrid system was used by our collaborator, Richard Furlanetto, to identify cDNAs in a human fetal brain library which encode proteins which interact with the autophosphorylated cytoplasmic domain of the IGF-I receptor. Two of these clones were identified as containing the SH2 domain of Grb10 and three clones represented two isoforms of 14-3-3 proteins. Grb10 was identified by Margolis by screening a mouse expression library with activated EGF receptor cytoplasmic domain whereas interaction of 14-3-3 proteins with a growth factor receptor was not previously described. We used the yeast two-hybrid system to map the domains of the cytoplasmic domain of the IGF-I receptor which interact with Grb10 and 14-3-3. 14-3-3 interacted fully with a receptor deleted at aa 1310 but did not interact with a receptor deleted at aa 1244, placing the interacting domain between aa 1244 and 1310. Mutation of tyrosines 1250 and 1251 within this domain to phenylalanine did not result in diminution of binding. Thus, although interaction of 14-3-3 with IGF-I receptor required an activated receptor, phosphorylation of tyrosines 1250 and 1251 is not required. Mutational analysis of IGF-I receptor binding to Grb10 indicated that the SH2 domain of Grb10 interacts with more than one phosphotyrosine containing motif in the receptor. Reports of in vivo experiments with cultured cells utilizing mutant constructs of the IGF-I receptor point to a trans mechanism for autophosphorylation of the receptor (two beta subunits within a heterotetramer phosphorylate each other). We examined the autophosphorylation of the cytoplasmic domain of the IGF-I receptor. IGF-I receptor cytoplasmic domain containing a histidine tail was expressed in Sf9 cells and purified on a metal affinity column. Autophosphorylation of the receptor was examined over a ten-fold range of concentration. Autophosphorylation was concentration independent consistent with an intramolecular phosphorylation mechanism. Surprisingly, we found evidence for a subpopulation of disulfide linked receptor dimers but were unable to identify dimers under the mild reducing conditions of the autophosphorylation assay.